The amyloid-ß1-42-oligomer interacting peptide D-AIP possesses favorable biostability, pharmacokinetics, and brain region distribution.

We have previously developed a unique 8-amino acid Aß42 oligomer-Interacting Peptide (AIP) as a novel anti-amyloid strategy for the treatment of Alzheimer's disease. Our lead candidate has successfully progressed from test tubes (i.e., in vitro characterization of protease-resistant D-AIP) to transgenic flies (i.e., in vivo rescue of human Aß42-mediated toxicity via D-AIP-supplemented food). In the present study, we examined D-AIP in terms of its stability in multiple biological matrices (i.e., ex-vivo mouse plasma, whole blood, and liver S9 fractions) using MALDI mass spectrometry, pharmacokinetics using a rapid and sensitive LC-MS method, and blood brain barrier (BBB) penetrance in WT C57LB/6 mice. D-AIP was found to be relatively stable over 3 h at 37 °C in all matrices tested. Finally, label-free MALDI imaging showed that orally administered D-AIP can readily penetrate the intact BBB in both male and female WT mice. Based upon the favorable stability, pharmacokinetics, and BBB penetration outcomes for orally administered D-AIP in WT mice, we then examined the effect of D-AIP on amyloid "seeding" in vitro (i.e., freshly monomerized versus preaggregated Aß42). Complementary biophysical assays (ThT, TEM, and MALDI-TOF MS) showed that D-AIP can directly interact with synthetic Aß42 aggregates to disrupt primary and/or secondary seeding events. Taken together, the unique mechanistic and desired therapeutic potential of our lead D-AIP candidate warrants further investigation, that is, testing of D-AIP efficacy on the altered amyloid/tau pathology in transgenic mouse models of Alzheimer's disease.

Cathepsin D: Analysis of its potential role as an amyloid beta degrading protease.

Proteolysis catalyzed by the major lysosomal aspartyl protease cathepsin-D (CTSD) appears to be of pivotal importance for proteostasis within the central nervous system and in neurodegeneration. Neuronal Ceroid Lipofuscinosis (NCL) type 10 is caused by a lack of CTSD leading to a defective autophagic flow and pathological accumulation of proteins. We previously demonstrated a therapeutic-relevant clearance of protein aggregates after dosing a NCL10 mouse model with recombinant human pro-cathepsin-D (proCTSD). Similar results could be achieved in cells and mice accumulating α-synuclein. Prompted by these positive effects and our in vitro findings showing that cathepsin-D can cleave the Alzheimer's Disease (AD)-causing amyloid beta peptides (Aß), we envisaged that such a treatment with proCTSD could similarly be effective in clearance of potentially toxic Aß species. We demonstrated that CTSD is able to cleave human Aß1-42 by using liquid chromatography-mass spectrometry. Intracerebral dosing of proCTSD in a NCL10 (CTSD knockout) mouse model revealed uptake and processing of CTSD to its mature and active form. However, the re-addition of CTSD did not obviously affect intracellular APP processing or the generation of soluble APP and Aß-species. ProCTSD treated HEK cells in comparison with untreated cells were found to contain comparable levels of soluble and membrane bound APP and Aß-species. Also, the early intracranial application (P1 and P20) of proCTSD in the 5xFAD mouse model did not change Aß pathology, plaque number and plaque composition and neuroinflammation, however we observed an increased level of Aß1-42 in the CSF. Our data confirm proteolytic cleavage of human Aß1-42 by CTSD but exclude a prominent role of CTSD in APP processing and Aß degradation in our in vitro and in vivo models.

Plasma Amyloid-Beta Levels in a Pre-Symptomatic Dutch-Type Hereditary Cerebral Amyloid Angiopathy Pedigree: A Cross-Sectional and Longitudinal Investigation.

Plasma amyloid-beta (Aß) has long been investigated as a blood biomarker candidate for Cerebral Amyloid Angiopathy (CAA), however previous findings have been inconsistent which could be attributed to the use of less sensitive assays. This study investigates plasma Aß alterations between pre-symptomatic Dutch-type hereditary CAA (D-CAA) mutation-carriers (MC) and non-carriers (NC) using two Aß measurement platforms. Seventeen pre-symptomatic members of a D-CAA pedigree were assembled and followed up 3-4 years later (NC = 8; MC = 9). Plasma Aß1-40 and Aß1-42 were cross-sectionally and longitudinally analysed at baseline (T1) and follow-up (T2) and were found to be lower in MCs compared to NCs, cross-sectionally after adjusting for covariates, at both T1(Aß1-40: p = 0.001; Aß1-42: p = 0.0004) and T2 (Aß1-40: p = 0.001; Aß1-42: p = 0.016) employing the Single Molecule Array (Simoa) platform, however no significant differences were observed using the xMAP platform. Further, pairwise longitudinal analyses of plasma Aß1-40 revealed decreased levels in MCs using data from the Simoa platform (p = 0.041) and pairwise longitudinal analyses of plasma Aß1-42 revealed decreased levels in MCs using data from the xMAP platform (p = 0.041). Findings from the Simoa platform suggest that plasma Aß may add value to a panel of biomarkers for the diagnosis of pre-symptomatic CAA, however, further validation studies in larger sample sets are required.

Label-free distribution of anti-amyloid D-AIP in Drosophila melanogaster: prevention of Aß42-induced toxicity without side effects in transgenic flies.

Soluble oligomers of the 42-amino acid amyloid beta (Aß42) peptide are highly toxic and suspected as the causative agent of synaptic dysfunction and neuronal loss in Alzheimer's disease (AD). Previously, we have shown that a small, D-amino acid Aß42-oligomer interacting peptide (D-AIP) can neutralize human Aß42-mediated toxicity using in vitro and cell-based assays. In the present longitudinal study using a transgenic Drosophila melanogaster model, advanced live confocal imaging and mass spectrometry imaging (MALDI-MSI) showed that the eight amino acid D-AIP can attenuate Aß42-induced toxicity in vivo. By separating male and female flies into distinct groups, the resultant distribution of ingested D-AIP was different between the sexes. The Aß42-induced 'rough eye' phenotype could be rescued in the female transgenics, likely because of the co-localization of D-AIP with human Aß42 in the female fly heads. Interestingly, the phenotype could not be rescued in the male transgenics, likely because of the co-localization of D-AIP with a confounding male-specific sex peptide (Acp70A candidate in MSI spectra) in the gut of the male flies. As a novel, more cost-effective strategy to prevent toxic amyloid formation during the early stages of AD (i.e. neutralization of toxic low-order Aß42 oligomers without creating larger aggregates in the process), our longitudinal study establishes that D-AIP is a stable and highly effective neutralizer of toxic Aß42 peptides in vivo. Cover Image for this issue: doi: 10.1111/jnc.14512.

Hyperbranched Polyglycerol Derivatives as Prospective Copper Nanotransporter Candidates.

Hyperbranched polyglycerol (hPG) has been used as a multivalent scaffold to develop a series of nanocarriers capable of high-affinity encapsulation of copper (Cu). A rationally selected set of Cu-complexing motifs has been conjugated to hPG hydroxyl groups to render the constructs potentially usable as exogenous sources of Cu for addressing different pathological conditions associated with Cu-deficiency. We have utilized a newly discovered route to attach Cu-binding domains exclusively within a hPG core by selective differentiation between the primary and secondary hydroxyl groups of the polyol. These hPG-derivatives were found to form a stable complex with Cu ions depending on the type of immobilized ligands and corresponding degree of functionalization. In addition, these Cu-bearing nano-complexes demonstrated moderately cationic surface charge resulting in adjustable protein-binding characteristics and low cellular toxicity profile. We envision that these Cu-loaded hPG nanocarriers can be used as a stable platform to transport the metal ion across the systemic circulation to supply bioavailable quantity of Cu in disease-afflicted tissues.

Amyloid Precursor Protein (APP) Metabolites APP Intracellular Fragment (AICD), Aß42, and Tau in Nuclear Roles.

Amyloid precursor protein (APP) metabolites (amyloid-ß (Aß) peptides) and Tau are the main components of senile plaques and neurofibrillary tangles, the two histopathological hallmarks of Alzheimer disease. Consequently, intense research has focused upon deciphering their physiological roles to understand their altered state in Alzheimer disease pathophysiology. Recently, the impact of APP metabolites (APP intracellular fragment (AICD) and Aß) and Tau on the nucleus has emerged as an important, new topic. Here we discuss (i) how AICD, Aß, and Tau reach the nucleus and how AICD and Aß control protein expression at the transcriptional level, (ii) post-translational modifications of AICD, Aß, and Tau, and (iii) what these three molecules have in common.

Amyloid precursor-like protein 1 (APLP1) exhibits stronger zinc-dependent neuronal adhesion than amyloid precursor protein and APLP2.

The amyloid precursor protein (APP) and its paralogs, amyloid precursor-like protein 1 (APLP1) and APLP2, are metalloproteins with a putative role both in synaptogenesis and in maintaining synapse structure. Here, we studied the effect of zinc on membrane localization, adhesion, and secretase cleavage of APP, APLP1, and APLP2 in cell culture and rat neurons. For this, we employed live-cell microscopy techniques, a microcontact printing adhesion assay and ELISA for protein detection in cell culture supernatants. We report that zinc induces the multimerization of proteins of the amyloid precursor protein family and enriches them at cellular adhesion sites. Thus, zinc facilitates the formation of de novo APP and APLP1 containing adhesion complexes, whereas it does not have such influence on APLP2. Furthermore, zinc-binding prevented cleavage of APP and APLPs by extracellular secretases. In conclusion, the complexation of zinc modulates neuronal functions of APP and APLPs by (i) regulating formation of adhesion complexes, most prominently for APLP1, and (ii) by reducing the concentrations of neurotrophic soluble APP/APLP ectodomains. Earlier studies suggest a function of the amyloid precursor protein (APP) family proteins in neuronal adhesion. We report here that adhesive function of these proteins is tightly regulated by zinc, most prominently for amyloid precursor-like protein 1 (APLP1). Zinc-mediated APLP1 multimerization, which induced formation of new neuronal contacts and decreased APLP1 shedding. This suggests that APLP1 could function as a zinc receptor processing zinc signals to stabilized or new neuronal contacts.

Impact of amyloid precursor protein hydrophilic transmembrane residues on amyloid-beta generation.

Amyloid-ß (Aß) peptides are likely the molecular cause of neurodegeneration observed in Alzheimer's disease. In the brain, Aß42 and Aß40 are toxic and the most important proteolytic fragments generated through sequential processing of the amyloid precursor protein (APP) by ß- and γ-secretases. Impeding the generation of Aß42 and Aß40 is thus considered as a promising strategy to prevent Alzheimer's disease. We therefore wanted to determine key parameters of the APP transmembrane sequence enabling production of these Aß species. Here we show that the hydrophilicity of amino acid residues G33, T43, and T48 critically determines the generation of Aß42 and Aß40 peptides (amino acid numbering according to Aß nomenclature starting with aspartic acid 1). First, we performed a comprehensive mutational analysis of glycine residue G33 positioned within the N-terminal half of the APP transmembrane sequence by exchanging it against the 19 other amino acids. We found that hydrophilicity of the residue at position 33 positively correlated with Aß42 and Aß40 generation. Second, we analyzed two threonine residues at positions T43 and T48 in the C-terminal half of the APP-transmembrane sequence. Replacement of single threonine residues by hydrophobic valines inversely affected Aß42 and Aß40 generation. We observed that threonine mutants affected the initial γ-secretase cut, which is associated with levels of Aß42 or Aß40. Overall, hydrophilic residues of the APP transmembrane sequence decide on the exact initial γ-cut and the amounts of Aß42 and Aß40.

Nuclear translocation uncovers the amyloid peptide Aß42 as a regulator of gene transcription.

Although soluble species of the amyloid-ß peptide Aß42 correlate with disease symptoms in Alzheimer disease, little is known about the biological activities of amyloid-ß (Aß). Here, we show that Aß peptides varying in lengths from 38 to 43 amino acids are internalized by cultured neuroblastoma cells and can be found in the nucleus. By three independent methods, we demonstrate direct detection of nuclear Aß42 as follows: (i) biochemical analysis of nuclear fractions; (ii) detection of biotin-labeled Aß in living cells by confocal laser scanning microscopy; and (iii) transmission electron microscopy of Aß in cultured cells, as well as brain tissue of wild-type and transgenic APPPS1 mice (overexpression of amyloid precursor protein and presenilin 1 with Swedish and L166P mutations, respectively). Also, this study details a novel role for Aß42 in nuclear signaling, distinct from the amyloid precursor protein intracellular domain. Chromatin immunoprecipitation showed that Aß42 specifically interacts as a repressor of gene transcription with LRP1 and KAI1 promoters. By quantitative RT-PCR, we confirmed that mRNA levels of the examined candidate genes were exclusively decreased by the potentially neurotoxic Aß42 wild-type peptide. Shorter peptides (Aß38 or Aß40) and other longer peptides (nontoxic Aß42 G33A substitution or Aß43) did not affect mRNA levels. Overall, our data indicate that the nuclear translocation of Aß42 impacts gene regulation, and deleterious effects of Aß42 in Alzheimer disease pathogenesis may be influenced by altering the expression profiles of disease-modifying genes.

Novel zinc-binding site in the E2 domain regulates amyloid precursor-like protein 1 (APLP1) oligomerization.

The amyloid precursor protein (APP) and the APP-like proteins 1 and 2 (APLP1 and APLP2) are a family of multidomain transmembrane proteins possessing homo- and heterotypic contact sites in their ectodomains. We previously reported that divalent metal ions dictate the conformation of the extracellular APP E2 domain (Dahms, S. O., Könnig, I., Roeser, D., Gührs, K.-H., Mayer, M. C., Kaden, D., Multhaup, G., and Than, M. E. (2012) J. Mol. Biol. 416, 438-452), but unresolved is the nature and functional importance of metal ion binding to APLP1 and APLP2. We found here that zinc ions bound to APP and APLP1 E2 domains and mediated their oligomerization, whereas the APLP2 E2 domain interacted more weakly with zinc possessing a less surface-exposed zinc-binding site, and stayed monomeric. Copper ions bound to E2 domains of all three proteins. Fluorescence resonance energy transfer (FRET) analyses examined the effect of metal ion binding to APP and APLPs in the cellular context in real time. Zinc ions specifically induced APP and APLP1 oligomerization and forced APLP1 into multimeric clusters at the plasma membrane consistent with zinc concentrations in the blood and brain. The observed effects were mediated by a novel zinc-binding site within the APLP1 E2 domain as APLP1 deletion mutants revealed. Based upon its cellular localization and its dominant response to zinc ions, APLP1 is mainly affected by extracellular zinc among the APP family proteins. We conclude that zinc binding and APP/APLP oligomerization are intimately linked, and we propose that this represents a novel mechanism for regulating APP/APLP protein function at the molecular level.

Sulfated Hyperbranched and Linear Polyglycerols Modulate HMGB1 and Morphological Plasticity in Neural Cells.

The objective of this study was to establish if polyglycerols with sulfate or sialic acid functional groups interact with high mobility group box 1 (HMGB1), and if so, which polyglycerol could prevent loss of morphological plasticity in excitatory neurons in the hippocampus. Considering that HMGB1 binds to heparan sulfate and that heparan sulfate has structural similarities with dendritic polyglycerol sulfates (dPGS), we performed the experiments to show if polyglycerols can mimic heparin functions by addressing the following questions: (1) do dendritic and linear polyglycerols interact with the alarmin molecule HMGB1? (2) Does dPGS interaction with HMGB1 influence the redox status of HMGB1? (3) Can dPGS prevent the loss of dendritic spines in organotypic cultures challenged with lipopolysaccharide (LPS)? LPS plays a critical role in infections with Gram-negative bacteria and is commonly used to test candidate therapeutic agents for inflammation and endotoxemia. Pathologically high LPS concentrations and other stressful stimuli cause HMGB1 release and post-translational modifications. We hypothesized that (i) electrostatic interactions of hyperbranched and linear polysulfated polyglycerols with HMGB1 will likely involve sites similar to those of heparan sulfate. (ii) dPGS can normalize HMGB1 compartmentalization in microglia exposed to LPS and prevent dendritic spine loss in the excitatory hippocampal neurons. We performed immunocytochemistry and biochemical analyses combined with confocal microscopy to determine cellular and extracellular locations of HMGB1 and morphological plasticity. Our results suggest that dPGS interacts with HMGB1 similarly to heparan sulfate. Hyperbranched dPGS and linear sulfated polymers prevent dendritic spine loss in hippocampal excitatory neurons. MS/MS analyses reveal that dPGS-HMGB1 interactions result in fully oxidized HMGB1 at critical cysteine residues (Cys23, Cys45, and Cys106). Triply oxidized HMGB1 leads to the loss of its pro-inflammatory action and could participate in dPGS-mediated spine loss prevention. LPG-Sia exposure to HMGB1 results in the oxidation of Cys23 and Cys106 but does not normalize spine density.

Mechanisms of amyloid-ß34 generation indicate a pivotal role for BACE1 in amyloid homeostasis.

The beta­site amyloid precursor protein (APP) cleaving enzyme (BACE1) was discovered due to its "amyloidogenic" activity which contributes to the production of amyloid-beta (Aß) peptides. However, BACE1 also possesses an "amyloidolytic" activity, whereby it degrades longer Aß peptides into a non­toxic Aß34 intermediate. Here, we examine conditions that shift the equilibrium between BACE1 amyloidogenic and amyloidolytic activities by altering BACE1/APP ratios. In Alzheimer disease brain tissue, we found an association between elevated levels of BACE1 and Aß34. In mice, the deletion of one BACE1 gene copy reduced BACE1 amyloidolytic activity by ~ 50%. In cells, a stepwise increase of BACE1 but not APP expression promoted amyloidolytic cleavage resulting in dose-dependently increased Aß34 levels. At the cellular level, a mislocalization of surplus BACE1 caused a reduction in Aß34 levels. To align the role of γ-secretase in this pathway, we silenced Presenilin (PS) expression and identified PS2-γ-secretase as the main γ-secretase that generates Aß40 and Aß42 peptides serving as substrates for BACE1's amyloidolytic cleavage to generate Aß34.

Biophysical characterization as a tool to predict amyloidogenic and toxic properties of amyloid-ß42 peptides.

Amyloid-ß42 (Aß42) peptides are central to the amyloid pathology in Alzheimer's disease (AD). As biological mimetics, properties of synthetic Aß peptides usually vary between vendors and batches, thus impacting the reproducibility of experimental studies. Here, we tested recombinantly expressed Aß42 (Asp1 to Ala42) against synthetic Aß42 from different suppliers using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), circular dichroism (CD) spectroscopy, thioflavin T aggregation, surface plasmon resonance, and MTT cell viability assays. Overall, our recombinant Aß42 provided a reproducible mimetic of desired properties. Across experimental approaches, the combined detection of Aß42 dimers and random coil to ß-sheet transition only correlated with aggregation-prone and cytotoxic peptides. Conclusively, combining MALDI-MS with CD appears to provide a rapid, reliable means to predict the 'bioactivity' of Aß42.

Insights into the Impact of Gold Nanoclusters Au10SG10 on Human Microglia.

The purpose of the current study is to uncover the impact of small liganded gold nanoclusters with 10 gold atoms and 10 glutathione ligands (Au10SG10) on several biomarkers in human microglia. We established the links connecting the atomically precise structure of Au10SG10 with their properties and changes in several biomolecules under oxidative stress. Au10SG10 caused the loss of mitochondrial metabolic activity, increased lipid peroxidation and translocation of an alarmin molecule, high mobility group box 1 (HMGB1), from the nucleus to the cytosol. Molecular modeling provided an insight into the location of amino acid interaction sites with Au10SG10 and the nature of bonds participating in these interactions. We show that Au10SG10 can bind directly to the defined sites of reduced, oxidized, and acetylated HMGB1. Further studies with similar complementary approaches merging live-cell analyses, determination of biomarkers, and cell functions could lead to optimized gold nanoclusters best suited for diagnostic and bioimaging purposes in neuroscience.

Presymptomatic Dutch-Type Hereditary Cerebral Amyloid Angiopathy-Related Blood Metabolite Alterations.

BACKGROUND: Cerebral amyloid angiopathy (CAA) is one of the major causes of intracerebral hemorrhage and vascular dementia in older adults. Early diagnosis will provide clinicians with an opportunity to intervene early with suitable strategies, highlighting the importance of pre-symptomatic CAA biomarkers. OBJECTIVE: Investigation of pre-symptomatic CAA related blood metabolite alterations in Dutch-type hereditary CAA mutation carriers (D-CAA MCs). METHODS: Plasma metabolites were measured using mass-spectrometry (AbsoluteIDQ® p400 HR kit) and were compared between pre-symptomatic D-CAA MCs (n = 9) and non-carriers (D-CAA NCs, n = 8) from the same pedigree. Metabolites that survived correction for multiple comparisons were further compared between D-CAA MCs and additional control groups (cognitively unimpaired adults). RESULTS: 275 metabolites were measured in the plasma, 22 of which were observed to be significantly lower in theD-CAAMCs compared to D-CAA NCs, following adjustment for potential confounding factors age, sex, and APOE ε4 (p < 00.05). After adjusting for multiple comparisons, only spermidine remained significantly lower in theD-CAAMCscompared to theD-CAA NCs (p  < 0.00018). Plasma spermidine was also significantly lower in D-CAA MCs compared to the cognitively unimpaired young adult and older adult groups (p < 0.01). Spermidinewas also observed to correlate with CSF Aß40 (rs = 0.621, p = 0.024), CSF Aß42 (rs = 0.714, p = 0.006), and brain Aß load (rs = -0.527, p = 0.030). CONCLUSION: The current study provides pilot data on D-CAA linked metabolite signals, that also associated with Aß neuropathology and are involved in several biological pathways that have previously been linked to neurodegeneration and dementia.

The amyloid-ß degradation intermediate Aß34 is pericyte-associated and reduced in brain capillaries of patients with Alzheimer's disease.

An impairment of amyloid ß-peptide (Aß) clearance is suggested to play a key role in the pathogenesis of sporadic Alzheimer's disease (AD). Amyloid degradation is mediated by various mechanisms including fragmentation by enzymes like neprilysin, matrix metalloproteinases (MMPs) and a recently identified amyloidolytic activity of ß-site amyloid precursor protein cleaving enzyme 1 (BACE1). BACE1 cleavage of Aß40 and Aß42 results in the formation of a common Aß34 intermediate which was found elevated in cerebrospinal fluid levels of patients at the earliest disease stages. To further investigate the role of Aß34 as a marker for amyloid clearance in AD, we performed a systematic and comprehensive analysis of Aß34 immunoreactivity in hippocampal and cortical post-mortem brain tissue from AD patients and non-demented elderly individuals. In early Braak stages, Aß34 was predominantly detectable in a subset of brain capillaries associated with pericytes, while in later disease stages, in clinically diagnosed AD, this pericyte-associated Aß34 immunoreactivity was largely lost. Aß34 was also detected in isolated human cortical microvessels associated with brain pericytes and its levels correlated with Aß40, but not with Aß42 levels. Moreover, a significantly decreased Aß34/Aß40 ratio was observed in microvessels from AD patients in comparison to non-demented controls suggesting a reduced proteolytic degradation of Aß40 to Aß34 in AD. In line with the hypothesis that pericytes at the neurovascular unit are major producers of Aß34, biochemical studies in cultured human primary pericytes revealed a time and dose dependent increase of Aß34 levels upon treatment with recombinant Aß40 peptides while Aß34 production was impaired when Aß40 uptake was reduced or BACE1 activity was inhibited. Collectively, our findings indicate that Aß34 is generated by a novel BACE1-mediated Aß clearance pathway in pericytes of brain capillaries. As amyloid clearance is significantly reduced in AD, impairment of this pathway might be a major driver of the pathogenesis in sporadic AD.

Neurodegenerative Disease-Related Proteins within the Epidermal Layer of the Human Skin.

There is increasing evidence suggesting that amyloidogenic proteins might form deposits in non-neuronal tissues in neurodegenerative disorders such as Alzheimer's or Parkinson's diseases. However, the detection of these aggregation-prone proteins within the human skin has been controversial. Using immunohistochemistry (IHC) and mass spectrometry tissue imaging (MALDI-MSI), fresh frozen human skin samples were analyzed for the expression and localization of neurodegenerative disease-related proteins. While α-synuclein was detected throughout the epidermal layer of the auricular samples (IHC and MALDI-MSI), tau and Aß34 were also localized to the epidermal layer (IHC). In addition to Aß peptides of varying length (e.g., Aß40, Aß42, Aß34), we also were able to detect inflammatory markers within the same sample sets (e.g., thymosin ß-4, psoriasin). While previous literature has described α-synuclein in the nucleus of neurons (e.g., Parkinson's disease), our current detection of α-synuclein in the nucleus of skin cells is novel. Imaging of α-synuclein or tau revealed that their presence was similar between the young and old samples in our present study. Future work may reveal differences relevant for diagnosis between these proteins at the molecular level (e.g., age-dependent post-translational modifications). Our novel detection of Aß34 in human skin suggests that, just like in the brain, it may represent a stable intermediate of the Aß40 and Aß42 degradation pathway.

Amyloid Precursor Protein Dimerisation Reduces Neurite Outgrowth.

The amyloid precursor protein (APP) undergoes extensive metabolism, and its transport and proteolytic processing can be modulated by its ability to form a homodimer. We have investigated the functional consequences of stabilised APP dimer expression in cells by studying the engineered dimerisation of the APPL17C (residue 17 in Aß sequence) construct, which is associated with a 30% increase in APP dimer expression, on APP's neurite outgrowth promoting activity. Overexpression of APPL17C in SH-SY5Y cells decreased neurite outgrowth upon retinoic acid differentiation as compared to overexpressing APPWT cells. The APPL17C phenotype was rescued by replacing the APPL17C media with conditioned media from APPWT cells, indicating that the APPL17C mutant is impairing the secretion of a neuritogenic promoting factor. APPL17C had altered transport and was localised in the endoplasmic reticulum. Defining the molecular basis of the APPL17C phenotype showed that RhoA GTPase activity, a negative regulator of neurite outgrowth, was increased in APPL17C cells. RhoA activity was decreased after APPWT conditioned media rescue. Moreover, treatment with the RhoA inhibitor, Y27632, restored a wild-type morphology to the APPL17C cells. Small RNAseq analysis of APPL17C and APPWT cells identified several differentially expressed miRNAs relating to neurite outgrowth. Of these, miR-34a showed the greatest decrease in expression. Lentiviral-mediated overexpression of miR-34a rescued neurite outgrowth in APPL17C cells to APPWT levels and changed RhoA activation. This study has identified a novel link between APP dimerisation and its neuritogenic activity which is mediated by miR-34a expression.

Aß34 is a BACE1-derived degradation intermediate associated with amyloid clearance and Alzheimer's disease progression.

The beta-site APP cleaving enzyme 1 (BACE1) is known primarily for its initial cleavage of the amyloid precursor protein (APP), which ultimately leads to the generation of Aß peptides. Here, we provide evidence that altered BACE1 levels and activity impact the degradation of Aß40 and Aß42 into a common Aß34 intermediate. Using human cerebrospinal fluid (CSF) samples from the Amsterdam Dementia Cohort, we show that Aß34 is elevated in individuals with mild cognitive impairment who later progressed to dementia. Furthermore, Aß34 levels correlate with the overall Aß clearance rates in amyloid positive individuals. Using CSF samples from the PREVENT-AD cohort (cognitively normal individuals at risk for Alzheimer's disease), we further demonstrate that the Aß34/Aß42 ratio, representing Aß degradation and cortical deposition, associates with pre-clinical markers of neurodegeneration. We propose that Aß34 represents a marker of amyloid clearance and may be helpful for the characterization of Aß turnover in clinical samples.

APLP1 is endoproteolytically cleaved by γ-secretase without previous ectodomain shedding.

Regulated intramembrane proteolysis of the amyloid precursor protein (APP) and its homologs, the APP like proteins APLP1 and APLP2, is typically a two-step process, which is initiated by ectodomain-shedding of the substrates by α- or ß-secretases. Growing evidence, however, indicates that the cleavage process for APLP1 is different than for APP. Here, we describe that full-length APLP1, but not APP or APLP2, is uniquely cleaved by γ-secretase without previous ectodomain shedding. The new fragment, termed sAPLP1γ, was exclusively associated with APLP1, not APP, APLP2. We provide an exact molecular analysis showing that sAPLP1γ was uniquely generated by γ-secretase from full-length APLP1. Mass spectrometry analysis showed that the sAPLP1γ fragment and the longest Aß-like peptide share the C-terminus. This novel mechanism of γ-secretase action is consistent with an ϵ-cut based upon the nature of the reaction in APP. We further demonstrate that the APLP1 transmembrane sequence is the critical determinant for γ-shedding and release of full-length APLP1. Moreover, the APLP1 TMS is sufficient to convert larger type-I membrane proteins like APP into direct γ-secretase substrates. Taken together, the direct cleavage of APLP1 is a novel feature of the γ-secretase prompting a re-thinking of γ-secretase activity modulation as a therapeutic strategy for Alzheimer disease.